The extracellular matrix of fungal cells is referred to as the cell wall. The cell wall is composed of interlocked proteins and polysaccharides and serves as a buffer between the cell and its environment. In pathogenic fungi, the wall is critical for survival in the host. Enzymes involved in the regulation and assembly of the cell wall are therefore excellent targets for antifungal drugs. The spore wall of Saccharomyces cerevisiae is a complex structure that forms de novo during the process of sporulation. Assembly of the spore wall provides an excellent system to investigate both the process of fungal wall assembly and the signaling pathways that coordinate that process. The Gip1-Glc7 phosphatase complex mediates a signaling pathway that initiates assembly of the wall only after the completion of cytokinesis. A structure-function analysis of the Gip1 protein will be used to explore the basis by which membrane closure triggers Gip1-Glc7 signaling. The mechanism by which Gip1-Glc7 regulate a downstream component in the pathway, the transcription factor Gis1, will also be elucidated. Though the constituents of the fungal cell wall are known, the enzymes that assemble these materials into a functional wall are little understood. We have identified several genes encoding candidate enzymes involved in construction of the spore wall. These phenotypes of mutants in these genes will be characterized in detail. Additionally, the enzymatic activities of these proteins will be examined using an in vitro assembly assay. Components of the spore wall in S. cerevisiae are also found in the cell wall of the pathogen C. albicans where they are important for virulence of the organism. We will examine if these components are assembled into the C. albicans cell wall a similar fashion as in the S. cerevisiae spore wall. The remarkable ability of the spore wall to protect the spore from an array of environmental insults is due primarily to the outer layers of spore wall. These layers have two major constituents, chitosan, a glucosamine polysaccharide, and a unique polymer containing the crosslinked amino acid dityrosine. This dityrosine polymer is found on the outermost surface of the spore wall and acts in part as a diffusion barrier that limits the movement of soluble molecules into and out of the wall. To better understand how this polymer functions the structure of the dityrosine polymer will be determined using a combination of mass spectrometry and solid state 13C NMR. PUBLIC HEALTH RELEVANCE: Fungal cell walls act as a barrier between the fungal cell and its environment. The wall is essential for a fungal pathogen to evade the host immune response and antifungal drugs commonly act by interfering with wall synthesis. This study focuses on the assembly of the spore wall of baker's yeast, Saccharomyces cerevisiae, as a model for fungal wall assembly. Experiments are targeted towards understanding the regulation, assembly pathways, and structure of wall components.